DESCRIPTION: There is a critical need for improved in vitro human toxicology testing to screen and identify potential toxic compounds, the levels at which they are lethal, and evaluate their developmental toxicity on human neural stem cells (NSCs) at the cellular and molecular levels. In particular, understanding mechanisms underlying human toxicity and the role of compounds including environmental toxicants and drug candidates is one of major goals of federal agencies such as NIEHS, NIGMS, and EPA, and has important implications in human health and disease prevention. Although animal models and primary human cells have been extensively used for such toxicology studies, their use is limited by high species variability with little or no predictability of relevance to humans, the instability of primary cells, and insufficient supply of human primary cells including NSCs for high-throughput screening. Therefore, there is an urgent need to develop in vitro strategies to rapidly assess compound-induced toxicity, and accurately predict adverse responses in vivo. Using three-dimensional (3D) cultured, human NSCs, together with high-throughput methodology and high-content imaging (HCI), we propose to decipher the cellular and molecular mechanisms underlying the effects of toxic model compounds. The core hypotheses are: (i) miniaturized 3D NSC microarrays can maintain high neuronal cell functions by better mimicking in vivo microenvironments; (ii) blocking ion channels and transporters on NSCs can modulate cell differentiation and cytotoxicity; (iii) physiological in vivo effects of compounds and their mechanistic actions on NSCs could be replicated and elucidated in vitro via high-throughput, high- content cell function analysis; and (iv) such miniaturized 3D cell culture systems can be used to facilitate mechanistic toxicology assays, which in turn can improve predictability of toxicity in vivo. The specific aims of the proposed work are to (1) demonstrate high neuronal cell functions on 3D NSC microarrays within biomimetic microenvironments in high throughput on the chip, (2) investigate mechanistic actions of various classes of compounds on NSC microarrays using high-throughput, ion channel and transporter assays, and (3) establish HCI assays on 3D NSC microarrays to investigate mechanistic profiles of toxicity by compounds and their metabolites. This information is essential to better understand the mechanistic basis of pharmaceutical toxicology on embryonic and adult human cells and tissues, and prioritize environmental toxicants based on their potential adverse effects on humans.